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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48807

Title: 細胞黏附蛋白與紅血球發育
cell adhesion molecule and erythrocyte development
Authors: Chiu, Chao-Shuo
邱昭碩
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 血球生成;低氧誘發蛋白;造血微環境;細胞黏附蛋白;斑馬魚
Hematopoietic;hypoxia-inducible factor;hematopoietic microenvironment;cell adhesion molecule;zebrafish
Date: 2017
Issue Date: 2018-08-22T06:31:29Z
Abstract: 在脊椎動物中,紅血球是用來運送氧氣及養分的主要工具,在斑馬魚的胚胎中,前期的血球生成是在中間細胞團(ICM)進行,而後期的血球生成發生在主動脈-生殖腺-中腎(AGM),隨後遷移到中繼造血組織,如哺乳動物的胎肝(fetal liver)或是魚類的後端造血組織(CHT),最後再遷移至哺乳動物的骨髓或是魚類的腎髓質等永久性的造血組織。近年來的研究發現在哺乳類動物的骨髓中,造血微環境對造血細胞的增殖與分化相當重要。造血微環境是個複雜的結構,其中包含了支持性基質細胞(local supporting stromal cells)、胞外基質(extracellular matrix,ECM)、以及水溶性因子(soluble factors)等。近來的研究發現哺乳動物骨髓造血系統位於一特殊的低氧微環境,導致所在的細胞內低氧誘發蛋白(Hypoxia-inducible factors,HIFs)表現上升,其中HIF2α會促使血管細胞黏附分子(vascular cell-adhesion molecule 1,VCAM1)表現量上升,促進紅血球細胞進行分化作用。為探討魚類造血機制是否與哺乳動物相似,本實驗利用斑馬魚(Danio rerio)的胚胎造血系統作為模式,驗證硬骨魚胚胎的造血微環境是否亦呈現缺氧狀態,並探討造血相關細胞黏附分子VCAM1、PECAM1和以及Ang1-TEK細胞訊息系統是否接受低氧誘發蛋白的控制,參與斑馬魚胚胎血球細胞的分化作用。實驗結果發現在斑馬魚胚胎的第二期血球生成時期(36 hpf和48 hpf),其造血區域AGM確實呈現低氧的狀態,並在同處可偵測到HIF1α及HIF2α蛋白質的專一性表現。在基因表現方面,細胞黏附分子pecam1基因與血管內皮細胞受器tek/tie2基因均在胚胎的造血組織專一性表現,另一細胞黏附分子vcam1基因則並未表現在造血組織中,因此排除其參與胚胎造血作用的可能性。在血球生成的作用上, pecam1基因敲落會抑制初期紅血球的生成,但不影響後期紅血球生成。tek基因敲落亦明顯抑制了初期紅血球的生成。在有關與低氧誘發蛋白HIF1α的互動關係上,敲落hif1α明顯抑制了tek的表現,但不影響pecam1的表現,而敲落hif2α則不影響tek或是pecam1的表現。這些結果顯示PECAM1與Ang1-TEK訊息路徑參與了初期紅血球生成的作用,但是對後期紅血球的生成並無明顯的影響。其中tek基因的表現受到HIF1α的專一性的調控影響,但與哺乳動物不同的是pecam1並未受HIF1α的調控。本實驗結果顯示雖然魚類胚胎的造血區域與哺乳動物骨髓內均存在低氧的微環境,但是低氧誘發蛋白在斑馬魚胚胎內所參與的初期紅血球生成作用機制與哺乳動物骨髓造血系統並不相同。
There are two waves of hematopoiesis in zebrafish embryos. The primitive wave of hematopoietic production occurs in the intermediate cell mass (ICM) and the definitive wave hematopoiesis occurs in the aorta-gonad mesonephros (AGM). The new-formed HSCs first migrate to the caudal hematopoietic tissue (CHT) in the posterior region, and then migrate to the kidney marrow and thymus to generate all types of blood cells throughout their lifespan. Recent study found that a hematopoietic microenvironment is important in hematopoietic proliferation and differentiation of cells. Hematopoietic microenvironment is a complex structure, including local supporting stromal cells, extracellular matrix (ECM), and other soluble factors. In bone marrow, the hematopoietic microenvironment is located in a specific hypoxia environment and the VCAM1 expression in endothelial cells is regulated by HIF2α to promote hematopoietic differentiation. To investigate the mechanism of hematopoietic differentiation during development, here we use zebrafish as a model to study the status of hematopoietic microenvironment and the correlations between hypoxia inducible factors and cell adhesion molecules vcam1, pecam1, and tek in embryonic hematopoiesis. It appears that a hypoxic niches was formed in the hematopoietic regions at 36 hpf and 48 hpf stages of zebrafish embryos, which is associated with HIF1α and HIF2α expression. The genes encode cell adhesion molecule, PECAM1, and Angiopoietin 1 receptor, TEK/Tie2, are both expressed specifically in hematopoietic tissues. The gene encode Vcam1 is not transcribed in hematopoietic tissues. Apparently Vcam1 is probably not involved in the process of embryonic hematopoiesis. It was shown that knockdown of pecam1 inhibited primitive erythropoiesis, but did not have significant effects for definitive erythropoiesis. Knockdown of tek also blocked primitive erythropoiesis. Inhibition of hif1α decreased tek transcription and did not change pecam1 expression. It appears that knockdown of hif2α did not change tek and pecam1 transcription. These results suggest that cell adhesion molecule PECAM1 and Ang1-TEK signaling involves primitive erythropoiesis, and tek is specifically regulated by HIF1α. Nevertheless, pecam1 is not controlled by either hypoxia inducible factor-1α or -2α. It appears that a specific hypoxia niche is highly conserved in all vertebrate hematopoietic microenvironment. However, the mechanisms that hypoxia signaling mediated hematopoiesis in mammalian bone marrow and fish embryos are quite different.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G001043B023.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48807
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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